title: N- and O-glycosylation in the murine synaptosome.
abstract: We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.
authors.affiliation: Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, School of Pharmacy, University of California San Francisco, San Francisco, California 94158-2517;
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Trinidad
authors.forename: Ralf
authors.initials: R
authors.lastname: Schoepfer
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002240:Carbohydrate Sequence; D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid; D005643:Fucose / Q000737:chemistry* / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014909:Wheat Germ Agglutinins / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D009077:Mucins; D014909:Wheat Germ Agglutinins; D005643:Fucose; D008358:Mannose